Method of detecting a nucleic acid

ABSTRACT

Disclosed is a method of detecting specific nucleic acids using an oligonucleotide linked to a cleavable tag. The presence of a specific nucleic acid in a population of nucleic acids is determined by hybridizing an oligonucleotide containing the tag to a population of nucleic acids, separating hybridizing bound oligonucleotides, and then removing and identifying the tag. Also provided are compositions and kits comprising oligonucleotides linked to a cleavable tag.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e)(1) from U.S.Serial No. 60/072,643 (herein incorporated by reference), which wasfiled Jan. 27, 1998.

FIELD OF THE INVENTION

The present invention relates generally to the field of nucleic aciddetection and specifically to a method for identifying a specificnucleic acid in a population of nucleic acids using a cleavablemolecular tag linked to an oligonucleotide.

BACKGROUND OF THE INVENTION

An important part of functional genomics is the analysis of genetranscription in a large population of nucleic acids. Transcriptionanalysis can potentially allow for the determination the identity ofeach gene expressed in a cell and the relative amount of transcriptexpressed compared with a control sample. Such analyses can thus revealthe “gene state” of a cell type or organism. However, quantitativeanalyses of transcription levels in a cell can be limited by therelatively large amount of the input RNA sample material needed, and bythe wide variation in the abundance of different transcripts in apopulation of nucleic acid molecules, e.g., in the relative abundance ofRNA molecules present in a single cell. The relative abundance, ordynamic range, can vary about four logs (10,000-fold) in RNA frompopulations of homogeneous cells or single cell assays. The range mayvary for an additional three orders or more of magnitude forheterogenous cell samples.

There exists a need for a method detecting specific nucleic acidsequences in small amounts of a population of nucleic acid sequence, andwhose abundance can vary over several orders of magnitude.

SUMMARY OF THE INVENTION

The invention is based on the discovery that cleavable tags attached tooligonucleotides can be used to identify specific nucleic acids in apopulation or collection of nucleic acids. In the methods describedherein, tagged oligonucleotides are hybridized with a population orcollection of nucleic acids. Hybridized oligonucleotides are thenseparated from non-hybridized oligonucleotides, and the tag is cleavedfrom the hybridized oligonucleotide and its identity determined. Theidentity of the tag thus reveals the identity of the oligonucleotide andthe nucleic acid in the population or collection of nucleic acids towhich the oligonucleotide hybridized.

The invention provides methods of detecting a specific nucleic acid in apopulation or collection of nucleic acids. In other embodiments, theinvention provides compositions of tagged oligonucleotides and kitscomprising tagged oligonucleotides for detecting specific nucleic acidsequences.

Among the advantages of the invention is increased sensitivity indetecting specific nucleic acid sequences in small amounts of an inputpopulation or collection of nucleic acid sequences. Another advantage ofthe invention is the ability to detect nucleic acids whose relativeabundance may differ over several orders of magnitude in a nucleic acidsample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic drawing of tagged oligonucleotides having anucleic acid sequence, which is complementary to at least a portion of adesired target sequence, linked to a tag moiety via a cleavable linker.

FIG. 2 shows a schematic drawing of a method of detecting a specifictranscript using a detector oligonucleotide.

FIG. 3 shows a schematic drawing of a method of detecting a specifictranscript using a detector oligonucleotide and a selectoroligonucleotide. The detector oligonucleotide and selectoroligonucleotide are linked by a ligation reaction.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a rapid, quantitative process fordetecting a specific nucleic acid sequence in a population or collectionof nucleic acid sequences. The method is based on the use of a taggedoligonucleotide that is complementary to at least a portion of aspecific nucleic acid sequence in the population or collection ofnucleic acid whose presence or abundance is to be assessed.

Three tagged oligonucleotides according to the invention are illustratedin FIG. 1, where they are labeled detector primer 1, detector primer 2,and detector primer 3. Each detector primer includes a specifiedoligonucleotide sequence attached to tags 1-3, respectively, via acleavable linker (CL). Two of the detector primers shown havecomplementary sequences in a population of target RNAs.

The detection of specific nucleic acid sequences using detector primersis shown schematically in FIG. 2. In the first step, detector primers1-3 are mixed with a population of target RNAs. For each specificnucleic acid sequence to be detected, one or more selectoroligonucleotides are designed to specifically anneal with a desiredtarget nucleic acid.

Step 2 illustrates the results of annealing the detector primers to thetarget nucleic acids to form a mixture of hybridized and non-hybridizedoligonucleotides. In step 3, the hybridized detector oligonucleotidesare separated from the non-hybridized oligonucleotides. The presence ofthe polyA⁺-tract on target RNAs is used in this example to separatepolyA⁺-containing RNA molecules bound to the detection primers fromnon-hybridized detection primers and nucleic acids lacking a polyA⁺region.

The linker is cleaved in step 4 to release the tag from theoligonucleotide. In steps 5 and 6, the released tag is purified, ifnecessary, and its identity determined. In the example shown, the tag isidentified using mass spectrometry. The example illustrates that it isthe tag itself, rather than the oligonucleotide, that is ultimatelydetected. By identifying and quantitating the tags associated withprimers that anneal stably, as opposed to tags from oligonucleotidesthat do not anneal, it is possible to indirectly but quantitativelyscore the presence and amount of the target nucleic acid.

A single hybridization reaction will typically contain multipledifferent detector oligonucleotides corresponding to different targetnucleic acids. The different target sequences, as is explained in moredetail below, can be, for example, different RNAs or different sequenceswithin the same RNA. When the tag is detected using mass spectrometry,very high level multiplexing is possible because of the ability of themass spectrometry to discriminate tags differing from each other bysmall mass increments.

At the end of this process, it is possible to deduce whether aparticular nucleic acid sequence, e.g., an RNA sequence or DNA sequencewas present in the original sample collection of nucleic acids, e.g,.DNA or RNA from a cell lysate, RNA preparation, or other biologicalsample containing RNA or a nucleotide representation of the RNA such ascDNA, by the presence of the tag corresponding to the taggedoligonucleotide. The presence and the absolute and/or the relativeamount of a given tag reflects the amount of the target complementarynucleic acid present in the original sample.

The range of quantitation possible will depend on how the annealingreactions are constructed and executed, on the choice of tags and ondetails of the particular methodology used to distinguish the tags.Depending on the specific use, the design parameters can be varied tofavor diversity of target, sensitivity, and dynamic range. For example,multiple different oligonucleotides with distinct tags directed atdifferent parts of the same gene or RNA can be used as informativeinternal controls. Similarly, if the same tag is used for multipleoligonucleotides directed at the same target nucleic acid, one canelevate sensitivity, as for a rare target RNA. Thus, the presentinvention provides a way to detect specific target sequences using smallamount of starting material, and to examine levels of both rare andabundant nucleic acid sequences in a collection of nucleic acidsequences.

In a first embodiment, the invention provides a method for the detectionof a specific nucleic acid sequence, or target sequence, in acollection, or sample, of nucleic acid sequences. The collection ofnucleic acid sequences can include, e.g, DNA, RNA, or a mixture of DNAand RNA, and can be from a cell, either prokaryotic or eukaryotic, ornon-cellular agent such as a virus or viroid. The target source can bewithin a mRNA, hnRNA, rRNA, tRNA, or snRNA, or within DNA, e.g.,nuclear, mitochondrial, chloroplast DNAs, as well as plasmid DNAs.

The collection of nucleic acids can be from one source, or from one ormore sources, e.g., DNA from a single individual, a mixture of two ormore individuals, or a mixture of cell types from the same individual.Alternatively, the collection can include RNA from a particular cell,tissue, or cell extract, including at a particular physiological state,developmental stage, or in a particular disease extract.

The method includes providing at least one oligonucleotide, also termedthe “detector oligonucleotide”, covalently linked to a removable tag,which is also referred to herein as a “tagged oligonucleotide.” Theidentity of both the tag and oligonucleotide sequence to which thetagged attached is known.

The term “tag” as used herein refers to a chemical moiety that can bedetected and quantified. In preferred embodiments the tag is detectedand quantified using mass spectrometry. While the tag need not have aparticular type of chemical structure, it must be stable while thelinked oligonucleotide is hybridized to the target nucleotide sequence.Preferably, the tag is also stable upon long term storage and is linkedto the oligonucleotide so that its subsequent removal is simple,efficient, and can be occur without rendering the tag non-identifiable.Examples of tags include, e.g., amino acids, small peptides composed of2-20 amino acids, and polymers with different numbers of methyl groupsor other simple repeating units.

The term “oligonucleotide” as used herein refers to primers or oligomerfragments comprised of two or more deoxyribonucleotides orribonucleotides, preferably more than three. The exact size will dependon many factors, which in turn depend on the ultimate function or use ofthe oligonucleotide. In some embodiments, the oligonucleotide is, e.g.,8-50 nucleotides, 15-45 nucleotides, or 17-35 nucleotides in length.

The method also includes contacting the oligonucleotide with acollection of nucleic acid sequences under conditions which permithybridization of the oligonucleotide to a complementary sequence in thecollection of nucleic acid sequences, to form a mixture of hybridizedand non-hybridized oligonucleotides. The contacting step is typicallyconducted with the annealing of the tagged oligonucleotide with thecollection of nucleic acids under conditions of molar excess ofoligonucleotide to target, and other kinetic conditions such that theannealing is stopped near or beyond kinetic termination. Thehybridization reactions can be done in either solid phase or solution,but for many applications solution hybridization will be preferablebecause the reactions can readily be driven to kinetic completion and totarget saturation.

In some applications the oligonucleotide will have perfectcomplementary, or nearly perfect complementary, to a region of a targetsequence in a collection of nucleic acids. This will be desirable whenthe target sequence differs by only one or a few nucleotides in sequencefrom other sequences in a collection of nucleic acid sequences. Thissituation can arise, for example, when distinguishing two alleles of agene that may differ from one another in a single nucleotide sequence.For other applications, perfect complementary between theoligonucleotide sequence and a target sequence will not be necessary.

The method also comprises separating hybridized oligonucleotides fromnon-hybridized oligonucleotides. Separation can be done in any ofseveral methods and, depending on the desired level of sensitivity, canbe designed to be more or less stringent for eliminating nonhybridizedmaterial. Higher stringency conditions will generally be required if,for example, the tagged oligonucleotide sequence differs by only one ora few nucleotides from non-target sequences in the collection of nucleicacid sequence, e.g., when the tagged oligonucleotide is used todistinguish alleles of a gene which differ in single nucleotide. Higherstringency conditions will also be desirable when a small amount of thetarget nucleic acid is present, while lower stringency conditions may beacceptable when large amounts of the target nucleic acid is present, orwhen the signal to noise ratio is otherwise high.

While the method is not limited to a particular scheme for separatinghybridized and non-hybridized oligonucleotides, it is important that theseparation scheme be compatible with the chemical properties of thetags, i.e., the separation step must not alter the tag such that the tagcannot be identified subsequently.

In some embodiments, structural features found on some nucleic acidmolecules can be used to separate bound and unbound oligonucleotides.One such separation procedure based on detecting polyA⁺ sequences inmRNA and is shown schematically in FIG. 2. Most mRNAs in eukaryoticcells have a polyA⁺ tract at their 3′ end, and methods for purificationon the basis of the presence of polyA⁺ tract are well established andsimple. In this example, all RNA containing polyA⁺, as well as thosedetector oligonucleotides that are stably associated with RNA, areremoved from nonhybridized oligonucleotides because the hybridizedoligonucleotides co-enrich with the polyA⁺ RNA.

The separation can be further enhanced, with a resulting increase inpurification and downstream increase in signal to noise ratios, bycombining the poly A⁺ selection with another selection method, e.g., amethod based on recognizing 5′ cap structures on some eukaryotic mRNAs.Other methods of enhancing separation are by performing multipleiterations of the selection, or by using selection with a secondoligonucleotide termed the selector oligonucleotide, which is explainedin more detail below.

Also included in the method of the invention is removal of the tag fromthe hybridized oligonucleotides. Tags can be removed from theirrespective oligonucleotides by methods known in the art, e.g., byappropriate chemical (or photochemical) reaction (indicated by the arrowin FIG. 1).

The method also includes identification of the cleaved tag. The tag canbe identified by any of several methods, including mass spectra,emission spectra, absorption spectra, and antibody-binding. In preferredembodiments the tag is identified by mass spectrometry.

The mass spectrophotometric method can be, e.g., time-of-flight,quadrupole, magnetic sector or ion trap mass spectrometry.

Because the tag and oligonucleotide to which the tag is originallylinked is known, determining the identity of the detached tag makes itpossible to deduce whether a nucleic acid sequence complementary to theoligonucleotide sequence was present in the sample.

The number of nucleic acid sequences analyzed can be increased by usingat least two distinct tagged oligonucleotides, i.e., taggedoligonucleotides having distinguishable tags. These tags will typicallyalso be attached to oligonucleotides that have distinguishablesequences. Multiple tagged oligonucleotides makes it possible todetermine the relative amount of two target nucleic acid sequences bymeasuring the relative amounts of their corresponding tags. In general,the range of quantitation possible will depend on how the annealingreactions are performed, the choice of the mass tags, and on theparticular detection methodology used. Depending on the particulartarget, sensitivity required, or the dynamic range examined, it ispossible to detect or quantitate sequences corresponding to differentparts of a single nucleic acid molecule, e.g., of parts of a single RNAmolecule.

In general, multiple oligonucleotides with distinct tags complementaryto different parts of the same gene or RNA can be used as informativeinternal controls. Alternatively, if the same tag is used on multipleoligonucleotides, each of which is complementary to different regions ofthe same target sequence, then one tag can be used to identify a singletarget nucleic acid. The additive use of multiple taggedoligonucleotides can enhance detection of nucleic acids occurring in lowabundance in the target collection of nucleic acid molecules.Conversely, a small number of tagged oligonucleotides may be used toidentify a target sequence abundant in the target population. Alteringthe number of oligonucleotides is a way to minimized problems of initialsensitivity and undesirably high dynamic range. This method can also beused in other situations where there is a large difference in theabundance of two target sequences in a collection of nucleic acids. Forexample, the method can be used to measure the relative amounts ofintron versus exon sequence for a single gene, or to detect the relativeamount of 5′ end sequence versus 3′ end sequence of a particulartranscript, or to determine the relative abundance of one allele of agene compared to a second in a population.

Additional diversity in the tagged oligonucleotides can be generated byvarying the linkage between the tag and the oligonucleotide. Forexample, two tagged oligonucleotide families can be prepared using aphotoabile linker “x” or a photolabile linker “y. ” The linkers willdiffer in the wavelength at which they are cleaved. One set of taggedoligonucleotides can be constructed having tags a, b, and c, linked via“x” to oligonucleotides 1, 2, 3, respectively. A second set ofoligonucleotides is constructed having tags a, b, and c linked via “y”to oligonucleotides 4, 5, and 6. Liberation of tag a at the“x”-responsive wavelength reveals the presence of a nucleic acidcomplementary to oligonucleotide 1, while liberation of tag a followingirradiation at the “y”-responsive wavelength indicates the presence of anucleic acid homologous to the oligonucleotide 4. Thus, varying thelinkage between the oligonucleotide and the tag allows for a relativelysmall number of tags to be used to identify multiple nucleic acids.

In another embodiment of the invention, the first oligonucleotide, ordetector oligonucleotide, is provided along with a second taggedoligonucleotide, which is also referred to herein as the “selectoroligonucleotide.” The selector oligonucleotide is designed to hybridizespecifically with the target nucleic acid sequence at a specificallyselected position other than that of the detector oligonucleotide. Theselector oligonucleotide is chosen so that it can be joined to thedetector oligonucleotide directly in a subsequent ligation step, or sothat it can be used along with the detector oligonucleotide to create anamplification product requiring both the detector oligonucleotide andthe selector oligonucleotide.

The selector oligonucleotide thus acts to increase the specificity ofdetection for detector oligonucleotides that are correctly hybridized totheir intended target nucleic acids compared to any less specificinteractions of detector oligonucleotides with non-target RNAs, whichmight correspond to truly nonspecific background or might be members ofrelated but non-identical genes within a gene family. While it willoften be desirable to use a selector oligonucleotide will be mostpreferable when detecting a target nucleic acid in very small amounts ofstarting material, it can also be used when there are larger amounts ofstarting material.

Unlike the detector mass tags, however, the selector tag need not bedifferent for each target RNA or gene to be assayed. Instead, it can beuniversal for a large family, or library, of corresponding detectoroligonucleotides. Thus, in the simplest case only one selector tag willbe used for all target RNAs or genes in a reaction. However, it isreadily apparent that increasing the number of detector oligonucleotideswill increase the number of sequences detectable using selectoroligonucleotides. Moreover, diversity can be increased by using alimited number of selector tags in combination with a limited number ofdetector tags.

The precise position of the selector oligonucleotide relative to thedetector oligonucleotide, and its “sense” relative to the detectoroligonucleotide and the target nucleic acid, will depend on the precisechemical nature of the oligonucleotides (DNA or RNA or other) and onwhether a ligation reaction or a polymerization reaction is to be usedin the subsequent steps.

A selector oligonucleotide in addition offers a facile handle forretrieving and physically separating properly hybridized detectoroligonucleotides though their physical linkage to a companion selectoroligonucleotide, e.g., in using ligase chain reaction or polymerasechain reactions based on the selector oligonucleotide and detectoroligonucleotide.

The use of a selector primer along with a detector primer in a ligationreaction is shown schematically in FIG. 3. In FIG. 3, the target nucleicacid is a cDNA and the detector primers are DNA. However, any variationon DNA that can be used by DNA ligase can also be used as a substrate.Following hybridization to a cDNA with detector primers and withselector oligonucleotides, a reaction is performed on the mixture usingDNA ligase as the catalyst. Such ligation reactions result in formationof a covalent phosphodiester link between the 3′-most residue of oneoligo and the 5′-most residue of the adjacent annealed oligo orpolynucleotide. The absolute and highly precise requirement for theplacement of detector and selector oligonucleotides on the same targetcDNA is extremely powerful for improving specificity of the reaction.Thus, the selector and detector oligonucleotides must be preciselyadjacent relative to each other, and the nucleotides that are joined byligase must be correctly base paired with the target cDNA. Thisrequirement confers significant additional specificity to the detectionmethod, compared with the hybridization of a single detector oligo byitself.

It is understood that this method can be varied by employing otheroligonucleotide species, e.g., RNA or synthetic nucleic acids andcorresponding ligases.

In another embodiment, the selector oligonucleotide and detectoroligonucleotides are used as primers in a PCR reaction with a DNApolymerase, e.g., a thermostable DNA polymerase, to amplify a region inthe target nucleic acid to obtain an amplification product. An advantageof using selector oligonucleotides and PCR to amplify the signalobtained using the detector primers is the increase in signal to noiseratio by requiring annealing of the selector primer at specified nearbysites. In addition, the absolute signal obtained will also increase. Thelatter increase in signal will come, though, at some cost in loss ofquantitative fidelity.

Examples of selector oligonucleotides include; 1) biotinylating selectorprimers coupled with subsequent purification with avidin/strepavidin, or2) labeling the selection primer with digoxygenin with subsequentpurification with anti-digoxygenin affinity reagents, or 3) conjugatingselector primers with any other physical or molecular tag. In thesimplest case, the tags attached to the selection primers are generalfor all selection primers rather than specific for each target. However,for some applications, more than one family of selector oligonucleotidesmight be used in the same hybridization mix, either to act as aninternal standard or for the purpose of generating more than one familyof products for later detector tag analysis. i.e. multiplexing).

Any affinity scheme that allows physical isolation of selectionoligonucleotides from non hybridized oligonucleotides can be used.However, the specificity using the selector oligonucleotide is enhancedonly if the selector oligonucleotide and detector oligonucleotide areultimately co-purified, or if a sequences complementary to the twooligonucleotides is generated, e.g., as in PCR.

In some embodiments, the tag on the selector oligonucleotide can be afluorescent dye or dye-impregnated bead that would allow use of opticalsorting to identify tagged oligonucleotides.

In another embodiment, the present invention provides a kit useful fordetection of a specific nucleic acid in a collection of nucleic acids.The kit includes one or more containers comprising containing at leastone oligonucleotide sequence covalently linked to a removable tag.

In yet another embodiment, the invention provides an isolatedoligonucleotide composition comprising at least one oligonucleotidesequence covalently linked to a removable tag. The term “isolated” asused herein includes polynucleotides substantially free of other nucleicacids, proteins, lipids, carbohydrates or other materials with whichthey are naturally associated. cDNA is not naturally occurring as such,but rather is obtained via manipulation of a partially purifiednaturally occurring mRNA. Such compositions are useful for theidentification of specific nucleic acids in a collection of nucleic acidsequences, e.g., corresponding to an expressed gene in a cell, tissue orcell extract.

Preferably the tag is detectable by mass spectroscopy. The compositionin various embodiments may include 1 or 2 to 10, 10², 10³, 10⁴, 10⁵, or10⁶ or more different distinguishable tagged oligonucleotides. Invarious embodiments the composition includes about 1-10⁶, 10-10⁵,10²-10⁴, or 10³-10⁴ oligonucleotide sequences. It is understood that thesame tag be linked to distinct oligonucleotide sequences, provided thatwhen the same tag is linked to more than one distinct oligonucleotidesequence, each oligonucleotide linked to the tag can be distinguished bythe way in which the tag is attached to the oligonucleotide.

In some embodiments, at least a portion of the oligonucleotide sequenceis complementary to a region of a transcribed gene.

Also included in the invention are isolated nucleic acids which includea sequence complementary to the oligonucleotide sequence of a taggedoligonucleotide.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Forexample, the molecular tags may be attached to nucleic acids longer thanoligonucleotide, e.g., 100-1000 nucleotides in length, and used toidentify homologous target sequences in a collection of nucleic acids.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

5 1 31 RNA ARTIFICIAL TARGET RNA 1 1 caugguagua guacgguacc gucuacggua a31 2 30 RNA ARTIFICIAL TARGET RNA 2 2 uagcuuacac cuagggaccu auuucaggcu30 3 18 DNA ARTIFICIAL DETECTOR OLIGO 1 3 tagtcggtac cgtactac 18 4 18DNA ARTIFICIAL DETECTOR OLIGO 2 4 gaaataggtc cctaggtg 18 5 18 DNAARTIFICIAL DETECTOR OLIGO 3 5 cggtattagg gcccagca 18

What is claimed is:
 1. A method for detecting a first target nucleic acid molecule and a second target nucleic acid molecule in a collection of at least two nucleic acid molecules having different nucleotide sequences, the method comprising: providing a first oligonucleotide covalently linked to a first removable tag by a first photocleavable linker, and a second oligonucleotide covalently linked to a second removable tag by a second photocleavable linker, wherein the first and second photocleavable linkers are cleavable at different wavelengths; contacting the first oligonucleotide and the second oligonucleotide with the collection of nucleic acid molecules in a single reaction under conditions which permit hybridization of the first oligonucleotide and the second oligonucleotide to complementary sequences in the collection of nucleic acid molecules, thereby forming a mixture of hybridized and non-hybridized oligonucleotides; separating hybridized oligonucleotides from non-hybridized oligonucleotides; removing the first tag from hybridized first oligonucleotides by irradiating the mixture of hybridized and non-hybridized oligonucleotides at a first wavelength, thereby cleavinthe first photocleavable linker; identifying the first tag by its known chemical property, thereby detecting the first target nucleic acid molecule; removing the second tag from hybridized second oligonucleotides by irradiating the mixture of hybridized and non-hybridized oligonucleotides at a second wavelength, thereby cleaving the second photocleavable linker; and identifying the second tag by its known chemical property, thereby detecting the second target nucleic acid molecule in the collection of nucleic acid molecules.
 2. The method of claim 1, wherein the contacting step is performed in solution.
 3. The method of claim 1, wherein the contacting step is performed with the first oligonucleotide and the second oligonucleotide being attached to a solid support, or with the collection of nucleic acid molecules being attached to a solid support.
 4. The method of claim 1, wherein the first oligonucleotide is about 8 to about 50 nucleotides in length.
 5. The method of claim 1, wherein the first oligonucleotide is about 20 to about 40 nucleotides in length.
 6. The method of claim 1, wherein the collection of nucleic acid molecules is selected from the group consisting of RNA and cDNA.
 7. The method of claim 1, wherein the tag is identified from a method selected from the group consisting of mass spectra, emission spectra, absorption spectra, and antibody-binding.
 8. The method of claim 1, wherein the tag is identified using a mass spectrophotometric method selected from the group consisting of time-of-flight, quadrupole, magnetic sector and ion trap mass spectrometry.
 9. A kit useful for detection of the presence of a specific nucleic acid sequence in a collection of nucleic acid sequences, the kit comprising one or more containers comprising a first container containing a first isolated nucleic acid composition comprising a first oligonucleotide sequence covalently linked to a first removable tag by a first photocleavable linker and a second container containing a second oligonucleotide sequence covalently linked to a second removable tag by a second photocleavable linker, wherein the first and the second photocleavable linkers are cleavable at different wavelengths and a distinct correlation between the first photocleavable linker and the sequence of the first oligonucleotide is known, and a distinct correlation between the second photocleavable linker and the sequence of the second oligonucleotide is known.
 10. The method of claim 1, wherein the amount of the identified first tag determines quantitatively the presence of the first target nucleic acid molecule identified from the collection of nucleic acids.
 11. The method of claim 1, wherein the first removable tag is an amino acid, a peptide, a polymer, biotin, digoxygenin, a fluorescent dye or a dye impregnated bead.
 12. The method of claim 1, where the first tag is identified by a method selected from the group consisting of absorption spectra, antibody binding and optical sorting.
 13. The method of claim 1, wherein the first tag and the second tag are identical.
 14. The method of claim 1, wherein the first tag and the second tag are different.
 15. The method of claim 1, wherein the first oligonucleotide and the second oligonucleotide are complementary to a coding region of a transcribed gene.
 16. The method of claim 1, wherein the first oligonucleotide and the second oligonucleotide are complementary to a coding region of the same transcribed gene. 